Project Summary HIV-1 infection is a major global health problem. Although the effective combined antiretroviral therapy has prolonged the life span of HIV-1 infected individuals, damaging effects of HIV-1 infection persist in brain. HIV-1 infected people are now experiencing HIV-1 associated neurocognitive disorders (HAND), the prevalence of which is over 50% in the USA. Although widespread and devastating to those affected, the pathogenesis of HAND remains unclear. Interestingly, HAND pathology is not proportional to HIV-1 viral load, and HIV-1 virus does not infect neurons. Thus, neurotoxic effects are deemed to be resulted from HIV-1 viral proteins and inflammatory responses. Among HIV-1 viral proteins, HIV-1 transactivator of transcription (HIV-1 Tat) continues to be implicated as a causative factor in the pathogenesis of HAND; We and others have consistently shown that HIV-1 Tat induces microglia-mediated neuroinflammation, synaptic disruption, and neurotoxicity, but the underlying mechanisms are still elusive. The objective here is to determine a novel endolysosome-dependent mechanism whereby HIV-1 Tat elicits and contributes to the development of HAND. Our central hypothesis is that HIV-1 Tat disrupts the assembly of vacuolar ATPase, which in turn, elevates endolysosome pH, thereby inducing microglia dysfunction, synaptic disruption, and neuronal injury, and that reduction of endolysosome pH with the administration of ML-SA1 attenuates HIV-1 Tat-induced neuroinflammation and neuronal injury. Guided by our findings, this novel hypothesis will be tested by pursuing three specific aims. (1) Dissect mechanisms by which HIV-1 Tat disrupts synaptic integrity and induces neurotoxicity in primary cultured neurons. (2) Dissect mechanisms by which HIV-1 Tat induces microglia dysfunction in primary cultured microglia. (3) Determine, in vivo, mechanisms by which HIV-1 Tat induces neuroinflammation, synaptic disruption, and neurotoxicity. We are the first to show that HIV-1 Tat elevates endolysosome pH. We believe that elevation of endolysosome pH plays a critical role in microglia-mediated neuroinflammation, synaptic disruption and neurotoxicity as occur in HAND. We have discovered that ML-SA1 reduces endolysosome pH, which could then block HIV-1 Tat-induced elevation of endolysosome pH. Therefore, a major goal of this work is to determine if elevation of endolysosome pH is central to the development of neuroinflammation, synaptic disruption, and neurotoxicity. Therefore, results form the proposed work will provide a significant advancement in the pathogenesis of HAND, and may lead to the discovery of new effective therapeutic strategies against HAND and thereby reduce suffering of HIV-1 infected individuals.